Thousands of terpenes and terpenoid derivatives found throughout Nature are involved in diverse biosynthetic and metabolic pathways such as cholesterol biosynthesis in humans and paclitaxel (Taxol) synthesis in the Pacific yew. Notably, many terpenoids have been used as medicinal agents since times of antiquity due to their analgesic, antibiotic, and antifungal properties. In spite of the universal importance of this family of natural products for human health, it is remarkable that the three-dimensional structures of terpenoid cyclases have only recently been reported. Terpenoid cyclases (a.k.a. synthases) catalyze the specific cyclization of a common allylic pyrophosphate substrate, such as farnesyl diphosphate, into one of hundreds of possible products. The terpenoid cyclase plays a critical role as a template in "channeling" the precise substrate and intermediate conformations leading to the formation of one exclusive product. Thus, the terpenoid cyclases comprise an exciting class of biosynthetic enzymes from both the biological and the chemical perspectives, and here the principal investigator addresses the relative dearth of structural information on this enzyme class. In the current funding period, the principal investigator has determined the X-ray crystal structures of three sesquiterpene cyclases: pentalenene synthase, aristolochene synthase, and trichodiene synthase. He aims to build upon this foundation in the next funding period by dissecting detailed structure-function relationships in trichodiene synthase. Specifically, he will study site-specific variants with altered metal binding properties, and he will also study variants engineered to generate altered products. Additionally, he will determine the structures of enzyme-inhibitor complexes that will yield mechanistic inferences, i.e., "snapshots" of the reaction coordinate of cyclization. In order to broaden the knowledge of structure-function relationships in terpenoid cyclases, he will also study the monoterpene cyclase (+)-bornyl diphosphate synthase and the diterpene cyclase taxadiene synthase. The structures of these enzymes will provide the first clues regarding C1 0, Cl 5, or C20 isoprenoid substrate specificity in the greater family of terpenoid cyclases, all of which are predicted to exhibit the characteristic 'terpenoid synthase fold.